We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline-seven enzymatic steps upstream of codeinone-accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

Transgenic Haveria bidentis (a C, species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO, assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO, assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H,O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO, partial pressures. Carbon isotope discrimination was measured concurrently with C O , and H,O exchange on leaves of the control plants and T, progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C, species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO, concentration and CO, leakage from the bundle sheath; however, some down-regulation of the C, cycle also occurred.The C, photosynthetic pathway requires the coordinated functioning of mesophyll and bundle-sheath cells within a leaf. Reactions of the C, cycle concentrate CO, in the bundle-sheath cells for assimilation by Rubisco (Hatch, 1987). This enhances Rubisco carboxylation while at the same time inhibiting Rubisco oxygenation, thus reducing the amount of photorespiration. The energy cost of this C0,-concentrating mechanism is 2 mo1 of ATP per regeneration of 1 mo1 of the primary CO, acceptor PEP, and any CO, that leaks from the bundle sheath rather than being fixed by Rubisco reduces the efficiency of the concentrating mechanism (Hatch, 1987). The compartmentation of the photosynthetic process between mesophyll and bundlesheath cells has complicated the study of C, photosynthesis, and the biophysical characterization of the C0,-concentrating function of C, photosynthesis has been experimentally difficult. Many of the parameters, such as bundle-sheath CO, concentration or the leakiness (4) of the bundle sheath (defined as the fraction of CO, generated by

The opium poppy is a source of the pharmaceuticals codeine, morphine and their derived analgesics. Here we describe the initial characterization of the poppy mutant known as top1 (for 'thebaine oripavine poppy 1'), which accumulates the morphine and codeine precursors thebaine and oripavine and does not complete their biosynthesis into morphine and codeine. The original discovery of top1 stimulated a re-engineering of the opioid industry in the island state of Tasmania, which grows over 40% of the world's licit opiates, in order to produce thebaine and oripavine efficiently from morphine-free poppy crops to provide precursors for highly effective analgesics and for treatment of opioid addiction.

We have measured the discrimination against 13C during CO2 assimilation in Flaveria bidentis wild type plants and in transgenic Flaveria bidentis plants transformed (1) with an antisense RNA construct targeted to the nuclear encoded gene for the small subunit of Rubisco—these plants had reduced amounts of Rubisco, decreased CO2 assimilation rates and increased carbon isotope discrimination, which was also evident in the carbon isotope discrimination of leaf dry matter; and (2) transformed with the mature coding region of carbonic anhydrase, CA, from tobacco (Nicotiana tabacum) in the sense direction under the control of the cauliflower mosaic virus 35S promoter—these plants had slightly increased CA activity in the mesophyll as well as a 2–4-fold increase in CA activity in the bundle-sheath cells. The introduction of tobacco CA manifested itself by a reduction in CO2 assimilation rate and an increase in carbon isotope discrimination. We suggest that the increased carbon isotope discrimination is a result of increased bicarbonate leakage out of the bundle sheath.

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C 3 species, Rubisco operates in a low CO 2 environment, which is suboptimal for both catalysis and carbamylation. In C 4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO 2 atmosphere close to saturation. To explore the role of Rubisco activase in C 4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C 4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T 1 progeny required high CO 2 (.1 kPa) for growth. They had very low CO 2 assimilation rates at high light and ambient CO 2 , and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO 2 . The amount of Rubisco was similar to that of wild-type plants. Experiments with the T 1 progeny of these four primary transformants showed that CO 2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C 4 photosynthetic pathway.The C 4 photosynthetic pathway is a biochemical CO 2 -concentrating mechanism that provides elevated CO 2 partial pressure (pCO 2 ) at the site of Rubisco carboxylation in the bundle sheath. This suppresses photorespiration and allows Rubisco (EC 4.1.1.39) to operate close to its maximal rate, such that CO 2 assimilation in C 4 plants is effectively CO 2 saturated in air (Hatch, 1987). Similar to photosynthesis in C 3 species, there is a strong correlation between Rubisco content of leaves and CO 2 assimilation rate at high irradiance, demonstrating that Rubisco carboxylation is among the rate-limiting steps of C 4 photosynthesis (Usuda et al., 1984;Hunt et al., 1985;Sage et al., 1987;Furbank et al., 1996;von Caemmerer et al., 1997). For CO 2 fixation to take place, Rubisco's catalytic sites must first be activated. This requires the carbamylation of a Lys residue within Rubisco's catalytic sites to allow the binding of a Mg 21 (for review, see Andrews and Lorimer, 1987). Sugar phosphates can also bind to Rubisco catalytic sites, and this interferes with both the process of carbamylation and full activity of the carbamylated enzyme (Badger and Lorimer, 1981;Edmondson et al., 1990a;Zhu and Jensen, 1991). In vivo, the activation and maintenance of Rubisco activity are facilitated by a second protein called Rubisco activase. Activase was first identified by analysis of the rca mutant of Arabidopsis (Arabidopsis thaliana), which required high CO 2 fo...

Infectious virions of the insect RNA virus Helicoverpa armigera stunt virus (HaSV; Omegatetravirus, Tetraviridae) were assembled in cultured plant protoplasts of Nicotiana plumbaginifolia in the absence of detectable replication. Assembly of the virus, which has not been grown in cell culture, required cotransfection of a DNA plasmid expressing the HaSV capsid gene in combination with either genomic RNA or with DNA plasmids carrying the complete cDNAs to the two HaSV genomic RNAs. Each cDNA was placed under the control of the cauliflower mosaic virus 35S promoter and followed by a cis-acting ribozyme so that the resultant transcripts corresponded precisely to the two genomic RNAs. Protoplast assembly of infectious particles was confirmed by EM and bioassay of host insect larvae, which became diseased and produced virus particles confirmed as HaSV. Variant transcripts carrying nonviral sequences at either or both termini of the RNAs showed no infectivity, except for RNA2 carrying only a 3' terminal extension. No replication of HaSV in protoplasts was detected in pulse-labeling and blotting experiments. Insects showed less severe disease symptoms when fed protoplasts transfected with only the RNA1 and coat protein plasmids. The symptomatic larvae contained only RNA1 and failed to yield infectious progeny virus, suggesting that RNA1 is capable of self-replication. This novel plasmid-based system confirms that the reported sequence of HaSV represents an infective genome and establishes a procedure for the reverse genetics of a tetravirus.

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